The generation and expansion of human endothelial cells (ECs) from readily obtainable nonvascular cell sources has great therapeutic potential for revascularization of ischemic and injured organs. However, the cultivation and expansion of stable ECs to clinically relevant scales, while maintaining their angiogenic signature, has not been achieved. Human adult-derived ECs have limited expansion potential and senesce after a few passages. Similarly, ECs derived from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSC) have limited proliferative capacity and are phenotypically unstable, often drifting into other non-vascular lineages upon serial passaging (JAMES et al., Nat Biotechnol, 28: 161-166 (2010)). Human ECs derived from endothelial progenitor cells (EPCs) (LYDEN et al., Nat Med, 7, 1194-1201 (2001); RAFII et al., Nat Med 9: 702-712 (2003); RAFII et al., Nat Rev Cancer, 2: 826-835 (2002); JIN et al., Nat Med, 12: 557-567 (2006)) and their progeny, endothelial colony forming cells (ECFCs) have shown significant proliferative potential (INGRAM et al., Blood, 104: 2752-2760 (2004); YODER et al., Blood, 109: 1801-1809 (2007)) when grown in pooled platelet-rich plasma (REINISCH et al., Blood, 113: 6716-6725 (2009)). However, whether EPCs and ECFCs could maintain their vascular stability to enable propagation of these cells to clinical scale remains unknown. The short-comings of adult- and hESC-based strategies are likely attributable to an insufficient appreciation of the transcription factors and microenvironmental cues as well as culture conditions that are necessary for establishing and maintaining EC identity.
Several members of the E-twenty six (ETS)-family of transcription factors (TFs), including ETV2 (LEE et al., Cell stem cell, 2: 497-507 (2008); SUMANAS et al., Blood, 111: 4500-4510 (2008)), FLI1 (LIU et al., Current Bio. 18: 1234-1240 (2008)), and ERG (MCLAUGHLIN et al., Blood, 98: 3332-3339 (2001)) have been implicated in regulating vascular development and angiogenesis (DE VAL et al., Dev Cell, 16: 180-195 (2009); SATO et al., Cell Struct Funct, 26: 19-24 (2001)). These TFs directly regulate the expression of genes associated with EC development and function. Adult ECs constitutively express several ETS factors, such as FLI1, ERG (isoforms 1 and 2), ETS1, ETS2, Elf1, Elk1, VEZF and ETV6, while ETV2 is transiently expressed during embryonic development and is absent in adult ECs (KATAOKA et al., Blood, 118: 6975-6986 (2011); LELIEVRE et al., The International Journal Of Biochemistry & Cell Biology, 33: 391-407 (2001)). Although many of these TFs play key roles in vascular specification (LIU et al., Circ Res, 103: 1147-1154 (2008); PHAM et al., Dev Biol, 303: 772-783 (2007)), it is unknown whether these EC-specific TFs can also switch on endothelial genes in non-vascular cells.
Human amniotic fluid-derived cells (ACs) represent a potential source of non-vascular cells that could be amenable to reprogramming into authentic ECs. Freshly isolated ACs that are obtained from individuals with broad genetic and ethnic backgrounds, are routinely cultured from the amniotic fluid of mid-gestation human fetus for diagnostic purposes. They display high proliferative potential, can be HLA-typed, cryopreserved, and publically banked for clinical use. ACs can give rise to various cell types, including epithelial cells, mesenchymal cells and neural cells (DE COPPI et al., Nature biotechnology, 25: 100-106 (2007); Prusa and Hengstschlager, Med Sci Monit, RA253-7 (2002)). A rare subset of c-Kit+ (CD117+) ACs, which comprises 0.2 to 2% of the AC population, represents multipotent amniotic fluid stem cells (AFS) (ARNHOLD et al., Stem Cells Int 2011, 715341 (2011); DE COPPI et al., Nature biotechnology, 25: 100-106 (2007)) that can give rise to various cell types, including epithelial cells, mesenchymal cells and neural cells. While some of these c-Kit+ cells are believed to express the pluripotent Oct-4 gene (DE COPPI et al., Nature biotechnology, 25: 100-106 (2007); PRUSA et al., Hum Reprod, 18: 1489-1493 (2003)), it is unclear whether these cells are truly pluripotent or are primarily multipotent cells. Indeed, the majority of ACs are believed to be lineage-committed cells. Three subclasses of lineage-committed ACs have been identified: Epithelioid (E-type), amniotic fluid (AF-type) and fibroblastic (F-type) (BOSSOLASCO et al., Cell Res, 16: 329-336 (2006)). E-type ACs are speculated to originate from fetal skin, while F-type cells are derived from connective tissue (Gosden, 1983; Hoehn and Salk, 1982).
Whether ACs can give rise to vascular cells has been the subject of investigation. Incubation of naïve human ACs or pre-selected c-Kit+ ACs in angiogenic culture conditions has led to the generation and outgrowth EC-like cells that express a few EC-specific markers, including VE-Cadherin, VEGFR2 and CD31 and can remodel into tubule like-structures (ENAVIDES et al., Tissue Eng Part A, (2012); DE COPPI et al., Nature biotechnology, 25: 100-106 (2007); KONIG et al., Stem cells and development, 21: 1309-1320 (2012); HANG et al., Stem cells and development, 18: 1299-1308 (2009)). However, these EC-like cells have low proliferative potential, do not express the complete repertoire of mature EC genes, nor has it been verified that their original AC signature is erased. As such, these cells cannot be considered as authentic vascular endothelial cells.